Isoliquiritigenin regulates the circ_0002860/miR-431-5p/RAB9A axis to function as a tumor inhibitor in melanoma

Abstract Background: Isoliquiritigenin (ISL) presents antitumor effects against melanoma cells. It is known that various circular RNAs (circRNAs) are involved in the development of melanoma. Therefore, the present study aims to investigate the molecular mechanisms of ISL and circ_0002860. Methods: Circ_0002860, microRNA-431-5p (miR-431-5p) and member RAS oncogene family (RAB9A) were detected through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. Cell viability was examined via cell counting kit-8 assay. The proliferation ability was assessed using colony formation assay. Cell apoptosis and cell cycle were determined by flow cytometry. Transwell assay was used for detection of migration and invasion. Western blot was conducted for protein analysis. Target binding was confirmed via dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. In vivo research was performed through xenograft tumor assay. Results: Circ_0002860 was downregulated by ISL in melanoma cells. ISL-induced inhibitory effects on cell proliferation, cell cycle progression, migration and invasion were alleviated by circ_0002860 overexpression. MiR-431-5p was a target of circ_0002860. Circ_0002860 eliminated the ISL-induced tumor inhibition via sponging miR-431-5p in melanoma cells. Circ_0002860 elevated the RAB9A level by targeting miR-431-5p. The function of ISL was related to miR-431-5p/RAB9A axis in melanoma progression. Tumor growth was reduced by ISL in vivo through downregulating circ_0002860 to regulate miR-431-5p and RAB9A levels. Conclusion: The current data indicates that ISL suppressed cell malignant progression of melanoma via targeting the circ_0002860/miR-431-5p/RAB9A pathway.


Background
Melanoma is one of the most common type of skin cancer arising from pigment-producing melanocytes, with high incidence around the world [1]. The primary cutaneous melanomas can be cured with surgical excision, but those tumors with locoregional metastases have a major risk of recurrence [2,3]. Isoliquiritigenin (ISL) is an important compound of chalcone, which is isolated from the roots of licorice plants [4]. ISL exhibits various types of biological activities, such as antitumor, anti-oxidative and antiinflammatory [5]. A recent study has indicated that ISL promotes apoptosis and represses metastasis in melanoma [6]. Xiang et al. [7] discovered that ISL reduced cell growth in melanoma via targeting miR-301b/LRIG1 axis. However, the antitumor mechanism of ISL is not fully clear in melanoma.
Circular RNAs (circRNAs) are covalently closed-loop transcripts characterized by regulating gene expression via serving as sponges of microRNAs (miRNAs) [8,9]. CircRNAs play key roles in the pathogenesis of most skin diseases, including melanoma [10]. Luan et al. [11] declared that circRNA_0084043 contributed to the malignant development of melanoma by upregulating Snail through sponging miR-153-3p. The heat map analysis suggested that circ_0002860 was dysregulated with a high level in melanoma tissues [11]. The association of circ_0002860 with the function of ISL in melanoma has never been explored.
This research was conducted to investigate the relation of the tumor-inhibitory role of ISL with circ_0002860. The interaction between circ_0002860 or RAB9A and miR-431-5p was explored. The underlying mechanism of ISL with circ_0002860/miR-431-5p/RAB9A was unraveled in melanoma.

Human samples and cell lines
The melanoma tissues (n = 50) and normal paracancerous tissues (n = 50) were acquired from the First Affiliated Hospital of Hengyang Medical College, University of South China. The written informed consent files were provided by all melanoma patients. This study has approved by the Ethics Committee of the First Affiliated Hospital of Hengyang Medical College, University of South China (approval number: HN20190822). The collected samples were stored in liquid nitrogen until the extraction of RNA or protein.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay
RNA samples from tissues and cells were extracted via TRI Reagent (T9424, Sigma), followed by cDNA synthesis through ReverTra Ace® qPCR RT Kit (FSQ-101, Toyobo, Kita-Ku, Osaka, Japan) and level detection using SYBR ® Green Realtime PCR Master Mix (QPK-201, Toyobo). Then the levels were standardized to β-actin and U6, and the relative analysis was implemented using the 2 -∆∆Ct method [17]. Additionally, circ_0002860 stability was detected via RT-qPCR. Cells were incubated with 3 mg/mL Actinomycin D (Sigma) or total RNA was treated with 3 U/μg RNase R (GENESEED), then circ_0002860 and linear CFLAR levels were quantified by RT-qPCR. All primer sequences were provided in Table 1.

Cell counting kit-8 (CCK-8) assay
After ISL treatment and cell transfection for 24 h, melanoma cells were incubated with 10 μL/well CCK-8 solution (KGA317, KeyGen, Nanjing, China) at 37 °C. After 2 h, cell absorbance of 450 nm was tested through the microplate reader and the percentage of viable cells in total cells was calculated.

Colony formation assay
Five-hundred cells were added into the 12-well plates. Cells were cultured for 14 days, then the white colonies were fixated with 4% paraformaldehyde (Sigma) for 10 min and stained with 0.1% crystal violet (Sigma) for 15 min. Cell colonies were photographed using a digital camera and counted by Image J software (NIH, Bethesda, MD, USA).

Flow cytometry
Cell apoptosis was evaluated via apoptosis kit (KGA106, KeyGen). A875 and SK-MEL-1 cells (5 × 10 5 ) in 200 µL binding buffer reacted with 10 µL annexin V-fluorescein isothiocyanate (annexin V-FITC) and 10 µL propidium iodide (PI). After co-incubation for 20 min, cell status was detected through the flow cytometer (BD Biosciences, San Diego, CA, USA) and apoptosis rate was expressed as the ratio of apoptotic cells in total cells. Cell cycle was determined by Cell Cycle Detection Kit (KGA511, KeyGen). A875 and SK-MEL-1 cells (1 × 10 6 ) were centrifuged at 2000 rpm for 5 min and cell pellets were fastened with 500 µL 70% cold ethanol (Sigma) at 4 °C overnight. Then, cells were washed with 200 µL phosphate buffer solution (Gibco) and added with 500 µL PI/RNase A working solution for 45 min. The red fluorescence at excitation wavelength 488 nm was examined under the flow cytometer (BD Biosciences), and DNA content was analyzed in different phases (G1, S, G2, M).

Transwell assay
Transwell chamber (Corning Inc., Corning, NY, USA) was used for migration assay and chamber enveloped with matrigel (Corning Inc.) was applied to measure cell invasion. The lower chamber was added with 500 µL cell medium and the upper chamber was seeded with 1 × 10 5 cells. After incubation for 24 h, the moved cells were fixated with 4% paraformaldehyde (Sigma) and dyed with 0.1% crystal violet (Sigma) for 20 min. Cell images were acquired at 100 × magnification and cells were counted through the inverted microscope (Olympus, Tokyo, Japan).

RNA immunoprecipitation (RIP) assay
Magna RIP RNA-Binding Protein Immunoprecipitation Kit (RIP-12RXN, Sigma-Aldrich) was used for binding analysis between circ_0002860 and miR-431-5p. A875 and SK-MEL-1 cells were incubated with the antibody-conjugated magnetic beads at 4 °C overnight. Anti-immunoglobulin G (anti-IgG) was used as the negative control group of anti-argonaute-2 (anti-Ago2). Input group without incubation of magnetic beads acted as the positive group. Total RNA was extracted from the beads, followed by detecting the levels of circ_0002860 and miR-431-5p.

Animal experiment
After transfection of vector or oe-circ_0002860 for 48 h, A875 cells (2 × 10 6 ) were subcutaneously injected into BALB/c male nude mice (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China). When tumors reached 50-100 mm 3 , mice were intraperitoneally injected with 20 mg/kg ISL or PBS every two days. There were 5 mice in PBS+vector, ISL+vector or ISL+oe-circ_0002860 group. Tumor volume (length × width 2 × 0.5) was examined every week, and mice were euthanatized through flowing CO 2 after four weeks. Tumors were excised from mice and weighed. The gene levels were determined via RT-qPCR and western blot. The protein level of Ki67 (CST, #9027) was measured using Immunohistochemistry (IHC) assay [19]. All procedures for animals were ratified by the Animal Ethical Committee of the First Affiliated Hospital of Hengyang Medical College, University of South China (approval number: HN20190822).

Statistical analysis
The experiments were performed with three replicates, then data were revealed as the mean ± standard deviation and analyzed via SPSS 22.0 (SPSS Inc., Chicago, IL, USA). The group difference was calculated using Student's t-test or analysis of variance (ANOVA) followed by Tukey's test, and p < 0.05 showed a significant difference.
The linear CFLAR expression was markedly reduced while circ_0002860 was more resistant to the treatment of Actinomycin D (Figures 1C, 1D) and RNase R ( Figures 1E, 1F), suggesting the high stability of circ_0002860 in A875 and SK-MEL-1 cells. The level of circ_0002860 was significantly inhibited by ISL with different concentrations, compared to the control group ( Figures 1G, 1H). Circ_0002860 might be related to the function of ISL in melanoma.

ISL impeded proliferation, cell cycle progression, migration and invasion of melanoma cells via inhibiting circ_0002860
The overexpression of circ_0002860 was achieved by transfection of oe-circ_0002860, and the efficiency was great contrasted with vector transfection in A875 and SK-MEL-1 cells (Figure 2A). ISL suppressed cell viability in CCK-8 assay ( Figure 2B) and cell proliferation in colony formation assay ( Figure 2C), but oe-circ_0002860 eliminated these influences. Flow cytometry manifested that circ_0002860 overexpression attenuated the promotion of cell apoptosis ( Figure 2D) and inhibition of cell cycle progression (Figures 2E, 2F) caused by ISL. By performing the transwell assay, cell migration and invasion abilities were shown to be enhanced by transfection of oe-circ_0002860 in ISLtreated A875 and SK-MEL-1 cells (Figures 2G, 2H). The protein analysis of epithelial-mesenchymal transition (EMT) indicated that ISL-induced E-cadherin upregulation and N-cadherin or Vimentin downregulation were relieved after circ_0002860 was overexpressed (Figures 2I, 2J). Altogether, ISL inhibited the progression of melanoma cells by downregulating circ_0002860.

ISL reduced tumor growth of melanoma in vivo by targeting circ_0002860 to regulate the miR-431-5p/ RAB9A axis
To investigate the association of ISL with circ_0002860, xenograft tumor models were constructed in mice. Tumor images were shown in Figure 7A. The weight and volume of tumors were inhibited in ISL+vector group relative to PBS+vector group, then ISL-induced tumor growth inhibition was offset by circ_0002860 overexpression (Figures 7B, 7C). RT-qPCR manifested that circ_0002860 downregulation ( Figure 7D) and miR-431-5p upregulation ( Figure 7E) were caused by ISL treatment, while these effects were abated by circ_0002860 level upregulation in mice.

Discussion
ISL has been affirmed to inhibit the progression of human malignancies. For instance, cell proliferation was reduced and apoptosis was enhanced by ISL in nasopharyngeal carcinoma via affecting the miR-32/LATS2/Wnt axis [20]. ISL impeded breast cancer cell proliferation and metastasis via mediating   miR-374a/PTEN/Akt network [21]. Huang et al. [22] stated that ISL downregulated cyclinD1 to inactive PI3K/AKT pathway, thus repressing oncogenesis and migration of hepatocellular carcinoma cells. The inhibitory effects of ISL on melanoma cell growth and cell cycle progression, as well as the promoting influence on apoptosis, were affirmed in melanoma cells. As for cell metastasis, ISL has resulted in inhibition of cell migration and invasion. In addition, EMT-related protein detection also demonstrated that ISL reduced the motility ability of melanoma cells. Collectively, our results confirmed that ISL acted as a tumor inhibitor in melanoma. Increasing circRNAs were differentially expressed and participated in the malignant development of melanoma. Circ_0020710 was upregulated in melanoma and promoted tumor progression in vitro [23]. CircRNA_0016418 and circZNF609 accelerated the biological behaviors including migration, invasion and EMT in melanoma cells [24,25]. Herein, circ_0002860 level was shown to be significantly elevated in melanoma samples and cells. Interestingly, circ_0002860 was downregulated in ISL-treated melanoma cells and circ_0002860 upregulation attenuated the ISL-induced effects on cell processes. Therefore, the antitumor role of ISL in melanoma was associated with the inhibition of circ_0002860.
CircRNA/miRNA/mRNA axis has been reported in different types of human tumors. CircRNA_102209 facilitated cell growth and development in colorectal cancer via targeting miR-761 to increase the RIN1 expression [26]. CircRNA LPAR3 could interact with miR-198/MET axis to enhance the metastatic capacity of esophageal cancer cells [27]. Circ_0074026 upregulated the ERBB4 level by absorbing miR-1304, thereby promoting carcinogenesis in glioma cells [28]. The current data suggested that circ_0002860 was a miRNA sponge of miR-431-5p and the downstream target RAB9A was regulated by circ_0002860 through mediating miR-431-5p in melanoma cells. Furthermore, cell assays manifested that ISL inhibited the melanoma progression via regulating the circ_0002860/ miR-431-5p axis and miR-431-5p/RAB9A axis. Hence, ISL was considered to act as a tumor repressor by targeting circ_0002860 to modulate miR-431-5p/RAB9A axis. Meanwhile, animal assay validated that circ_0002860 could mitigate the ISL-induced tumor growth suppression by the regulation of miR-431-5p and RAB9A levels.
For the first time, the functional mechanism of ISL with circRNA/miRNA/mRNA axis was disclosed in melanoma. Nevertheless, this study still has some limitations. For example, ISL has been indicated to be associated with signaling pathways in tumors. Exploring the downstream pathways of circ_0002860/ miR-431-5p/RAB9A axis will be beneficial for the understanding of ISL-induced antitumor function. In addition, the regulation of circ_0002860 was partly achieved by miR-431-5p/RAB9A axis in alleviating the effects of ISL. Other miRNA/mRNA networks remain to be explored for circ_0002860.

Conclusion
In conclusion, ISL inhibited cell progression (proliferation, cell cycle progression, migration, invasion and EMT) and promoted apoptosis of melanoma via mediating the circ_0002860/miR-431-5p/RAB9A axis (Figure 8). Circ_0002860 might be used as a molecular target for ISL treatment in melanoma.